For the past three decades, Mycobacterium tuberculosis infections have declined in the United States, and it was thought that the disease would be virtually eliminated by the year 2000. However, the trend was reversed in 1985, and, as of 1992, an estimated 51,700 cases in excess of the 1984 projections have occurred in the United States (41). Even more disturbing has been the commensurate rise in the isolation of M. tuberculosis strains resistant to one or more of the antibiotics commonly used to treat the infection. While rapid detection methods based on nucleic acid amplification technologies have offered the promise of early detection of M. tuberculosis infection, to control this resurgent health problem these new diagnostic methods must be complemented by rapid tests for determining effective antibiotic therapy. The mechanisms of resistance to several of the front line drugs in M. tuberculosis have been identified and shown to be the result of mutations in key genes. In particular reports indicate that more than 95% of M. tuberculosis isolates exhibiting resistance to rifampin harbor specific mutations within a 69-bp region of the rpoB gene, which encodes the beta subunit of RNA polymerase. An opportunity exists, therefore, to create a diagnostic procedure based on the rapid detection of these genetic changes which lead to drug resistance. Because of these observations that genetic mutations play a significant role in the development of drug resistance in M. tuberculosis, methods which rapidly detect such mutations may play an important role in the clinical management of patients with tuberculosis. In this Phase I proposal, we will develop a model system for rapidly detecting mutations which lead to rifampin-resistance in M. tuberculosis using our proprietary Cleavase(R) Fragment Length Polymorphism (CFLPTM) technology. This work will form the foundation of genetic-based diagnostic tests for drug resistance in M. tuberculosis all of which will be based on our rapid, cost effective, and highly accurate CFLPTM technology. PROPOSED COMMERCIAL APPLICATION: We will develop a rapid, simple, cost effective assay for detecting rifampin-resistant M. tuberculosis based on our novel CFLPTM technology. The assay will be able to potentially detect at least 97% of all rifampin resistant isolates reported to date. In Phase II we will expand the capabilities of the CFLPTM technology to allow detection of mutations leading to isoniazid and streptomycin resistance. This technology will enable thousands of dollars of savings in patient care costs.